16. THE FUNCTIONAL GENOME

Question 1

*What is functional genomics?

Answer 1

• Functional genomics is the study of how genes & the intergenic regions are responsible for processes
• Looks at gene functions & interactions

Question 2

What does WGS & WEGS identify & why is it proof for pathogenesis?

Answer 2

• WGS & WEGS identifies candidate genes
• 15 -20,000 SNPs are reduced to several candidate genes
• The candidate genes are then checked for co-segregation amongst family members
• However, proof of pathogenesis is needed because the candidate genes aren’t causative. In vitro & in vivo methods can be used for proof of pathogenesis

Question 3

Name 5 in vitro techniques for studying gene function

Answer 3

• In vitro techniques study microorganisms or biological samples outside their biological context such as a test tube or petri dish
  1. Cell cultures
  2. ShRNA
  3. SiRNA
  4. IPSCs
  5. CRISPR (both in vivo & in vitro)

Question 4

How can blood or tissue biopsies show whether a genetic variant affects a protein?

Answer 4

• Blood or tissue biopsies can be used to show whether a genetic variant affects a protein
• A Western blot analysis of samples can be used for comparison & can reveal any effects on protein
• However, biopsies are invasive & painful so cell cultures are preferred. Some genes of interest also don’t show up in blood

Question 5

What is a cell culture?

Answer 5

• A cell culture involves removing cells from an organism or animal & subsequently growing them in manipulated conditions which are favourable

Question 6

What are the advantages of cell cultures?

Answer 6

+ Cell cultures are a cheap, rapid & reproducible model for studying cells
+ It’s a good alternative to using animal models as fewer animals are used & therefore less ethical issues
+ There are many commercially available tissue specific cell lines, so these can be used in cell cultures

Question 7

What are the disadvantages of cell cultures?

Answer 7

• Cells behave differently in a petri dish compared to inside the organism. It’s a 2D environment so some of the factors are missing
• Doesn’t stimulate the actual conditions inside an organism. E.g doesn’t consider signals from other tissues such as paracrine signals
• Cell cultures don’t provide any information about gene expression & function so we don’t know how the gene can affect other tissues

Question 8

What is ShRNA?

Answer 8

• Short-hairpin RNA is an artificial molecule of RNA that can be used to silence gene expression
• It is vector based

Question 9

How can ShRNA be used to knock out genes?

Answer 9

1. ShRNA can be packaged into a plasmid & the expression can be controlled by RNA Polymerase II
2. Once the RNA is transcribed, it can be transported out of the nucleus by the protein EXPORTIN 5
3. It is then cleaved by a DICER
4. The cleaved fragments then bind to RISC (RNA induced silencing complex)
5. Leads to gene silencing

Question 10

Give two ways in which the localisation of an encoded protein can be studied

Answer 10

1. ANTIBODY STAINING

2. TRANSFECT CELLS WITH GFP TAGGED MUTATED GENE OR GENE OF INTEREST - for visualisation

Question 11

What are IPSCs?

Answer 11

• Induced pluripotent stem cells are pluripotent stem cells that have been reprogrammed with TF from adult somatic cells

Question 12

What are the steps for producing IPSCs?

Answer 12

1. Take a skin biopsy
2. Fibroblasts grown in culture
3. Adult somatic cells can be reprogrammed with transcription factors
4. Adult somatic cells can de-differentiate into IPSCs
5. IPCs can be stimulated under conditions to differentiate into certain cells

Question 13

What is SiRNA?

Answer 13

• Short-interfering RNA molecules have a similar method to ShRNA
• But SiRNAs are chemically synthesised & aren’t vector based so doesn’t use a plasmid

Question 14

What is CRISPR & it’s steps?

Answer 14

• CRISPR can be carried out both in vivo & in vitro
• CRISPR = Clustered regularly interspaced short palindromic repeats
  1. Guide RNA binds to Cas9 forming a complex
  2. Cas9 then searches for a target sequence by binding to Protospacer adjacent motif (PAM - 2-3bp sequence downstream of guide RNA)
  3. Cleavage of target sequence leads to knockout of the gene
  4. Mutations can be introduced with Non-homologous end joining or Homology directed repair

Question 15

Name 4 in vivo techniques for studying gene function

Answer 15

1. Animal studies - mouse/zebrafish
2. Morpholinos
3. CRISPR
4. ENU screens

Question 16

Give 4 reasons why mice are used as the mammalian model for human genetics?

Answer 16

+ Mice have an accelerated lifespan where 30 years = 1 human year, so their whole lifespan can be studied in a few years
+ Mice are small & reproduce quickly so they are easy to study
+ Mice are genetically similar to humans so they are likely to be affected by similar diseases
+ Using mice is more ethical than using larger animals or non-human primates

Question 17

How can mice be used to introduce mutations?

Answer 17

• A targetting vector can be constructed & inserted into the nucleus of embryonic pluripotent stem cells
• The targetting vector will contain a DNA cassette which is flanked upstream or downstream producing homology arms
• The casette replaces the knockout gene & the cells can be grown to blastocysts which can then be implanted into mice

Question 18

*Give three reasons for why zebrafish are a good human model for human genetics?

Answer 18

1. Zebrafish are cheap to use
2. Zebrafish develop in 5 days
3. Zebrafish produce many eggs which are optically transparent & they grow outside the mother’s body (ex vivo), making it easy to study them

Question 19

How can morpholinos be used to knockout genes in zebra fish?

Answer 19

• Morpholinos (MO) are nucleotide analogs that recognise & bind to short sequences at the transcription or splicing site

Question 20

What are two types of morpholinos?

Answer 20

1. TRANSLATION BLOCKING MOs - target the transcription site

2. SPLICE INHIBITING MOs - target the splice site